Method for screening compounds to identify beta-amyloid production modulators

ABSTRACT

The present invention is directed generally to methods and composition for monitoring the processing of epitope-tagged beta-APP. More specifically, the present invention relates to the use of such methods and composition for monitoring responses of cells expressing such epitope-tagged beta-APP or fragments thereof or cell free systems containing the epitope-tagged polypeptides to therapy of diseases associated with an altered metabolism of the beta amyloid precursor protein (APP), and for screening and evaluation of potential drugs for the treatment of these disorders, including Alzheimer&#39;s disease (AD).

This application claims the benefit of U.S. Provisional Application No.60/115,749 filed on Jan. 13, 1999.

FIELD OF THE INVENTION

This invention is directed to CDNA sequences, cell lines, andpolypeptides containing epitope(s) for high-affinity antibodies withinthe A-beta fragment of the beta-amyloid precursor protein (beta-APP). Apreferred embodiment of the invention relates to assays useful foridentifying and characterizing beta amyloid precursor processinginhibitors, and for purifying APP processing enzymes. Such assays areuseful in developing beta-APP processing modulators for the treatment ofdiseases associated with altered metabolism of beta-APP and anaccumulation of amyloid A-beta.

BACKGROUND OF THE INVENTION

The present invention is directed generally to methods and compositionsfor monitoring the processing of epitope-tagged beta-APP. Morespecifically, the present invention relates to the use of such methodsand compositions for monitoring responses of cells expressing suchepitope-tagged beta-APP, or fragments thereof. The present inventionalso relates to cell free systems containing the epitope-taggedpolypeptides, therapy of diseases associated with an altered metabolismof the beta-amyloid precursor protein (beta-APP), and for screening andevaluation of potential drugs for the treatment of these disorders,including Alzheimer's disease.

Alzheimer's disease (AD) is a degenerative brain disorder characterizedclinically by progressive loss of memory, temporal and localorientation, cognition, reasoning, judgment and emotional stability. ADis a common cause of progressive dementia in humans and is believed tobe one of the major causes of death in the United States. AD has beenobserved in all races and ethnic groups worldwide and presents a majorpresent and future health problem. No treatment that effectivelyprevents AD, or reverses the clinical symptoms and underlyingpathophysiology is currently known.

Histopathological examination of brain tissue obtained upon autopsy, orfrom neurosurgical specimens in affected individuals, revealed theoccurrence of amyloid plaques and neurofibrillar tangles in the cerebralcortex of such patients. Similar alterations were observed in patientswith Trisomy 21 (Down's syndrome), and hereditary cerebral hemorrhagewith amyloidosis of the Dutch-type. Neurofibrillar tangles arenonmembrane-bound bundles of abnormal proteinaceous filaments.Biochemical and immunochemical studies led to the conclusion that theirprinciple protein subunit is an altered phosphorylated from of the tauprotein (reviewed in Selkoe, D. J. Annu Rev Cell Biol 1994 10: 373-403).

Biochemical and immunological studies show that the dominantproteinaceous component of the amyloid plaque is a 4.2 kilodalton (kD)protein of from about 39 to about 43 amino acids in length. This proteinwas designated A-beta, beta-amyloid peptide, or beta/A4. In addition toits deposition in amyloid plaques, A-beta is also found in the walls ofmeningeal and parenchymal arterioles, small arteries, capillaries, andoccasionally venules. A-beta was first purified and a partial amino acidsequence reported in 1984 (Glenner and Wong, Biochem. Biophys. Res.Commun. 120: 885-890). The isolation and sequence data for the first 28amino acids are described in U.S. Pat. No. 4,666,829, the contents ofwhich are herein incorporated by reference in their entirety.

Compelling evidence accumulated during the last decade revealed thatA-beta is an internal polypeptide derived from a type 1 integralmembrane protein, termed beta-APP. Beta-APP is normally produced by manycells both in vivo and in cultured cells derived from various animalsand humans. A-beta is derived from cleavage of beta-APP by an as yetunknown enzyme (protease) system(s), collectively termed secretases. Theexistence of at least three proteolytic activities has been postulated.They include (a) beta secretase(s), generating the N-terminus of A-beta,(b) alpha secretase(s) cleaving in the region of the 16/17 peptide bondin A-beta, and (c) gamma secretases, generating C-terminal A-betafragments ending at position numbers 38, 39, 40, 42, and 43. The precisebiochemical mechanism by which A-beta is derived from beta-APP, and howit subsequently accumulates in cerebral tissue and blood vessels, iscurrently unknown.

Several lines of evidence suggest that the abnormal accumulation ofA-beta plays a key role in the pathogenesis of AD. First, A-beta is themajor protein found in amyloid plaques (Glenner G G, Wong, C W 1984Biochem Biophys Res Commun 120: 885-90). Second, A-beta is neurotoxicand may be causally related to neuronal death observed in AD patients(Pike C J, Burdick, D, Walencewicz A J, Glabe C G, Cotman C W 1993 JNeurosci 13: 1676-87). Third, missense DNA mutations at position 717 inthe 770 isoform of beta-APP can be found in affected members but not inunaffected members of several families with a genetically determined(familial) form of AD (Goate A, Chartier-Harlin M-C, Mullan M, Brown J,Crawford F et al 1991 Nature 349: 704-6). In addition, several otherbeta-APP mutations have been described in familial forms of AD (reviewedin Selkoe D J 1994 Annu Rev Cell Biol 1994 10: 373-403). Fourth, similarneuropathological changes have been observed in transgenic animalsoverexpressing mutant forms of human beta-APP (Hsiao K, Chapman P,Nilsen S, Eckman C, Harigaya Y, Younkin S, Yang F, Cole G 1996 Nature274: 99-102). Finally, individuals with Down's syndrome have anincreased gene dosage of beta-APP and develop early-onset AD. Takentogether, these observations strongly suggest that A-beta depositionsmay be causally related to Alzheimer's disease.

While large progress has been made in understanding the underlyingcause(s) of AD and other A-beta related diseases, there remains a needto develop methods and compositions for the treatment of the disease(s).Treatment methods could be based on compounds that inhibit the formationof A-beta in vivo. To identify and characterize such compounds,high-throughput screening methods are required to identify compoundsthat affect beta-APP processing. Specific assays for A-beta detectionshould be able to detect A-beta in biological samples at very lowconcentrations, as well as distinguishing between A-beta and otherfragments of beta-APP that may be present in the sample.

U.S. Pat. No. 4,666,829, suggests the use of an antibody to the 28-aminoacid fragment of A-beta to detect “Alzheimer's Amyloid Polypeptide” inbiological samples. This suggestion was not used in the presentinvention.

Several attempts to measure A-beta in biological samples byimmunological methods have been reported. While these studies detectvery low levels of A-beta peptides, no attempts to purify andcharacterize this immunoreactivity further and to determine whether itindeed represents A-beta have been reported.

U.S. Pat. No. 5,593,846 describes assays aimed at determining A-betalevels in biological samples using antibodies based on the native A-betasequence. No attempt was made to differentiate between A-beta peptideswith heterogenous C-termini by either enzyme-linked immunosorbant assay(ELISA), or radioimmunoassay (RIA). More specifically, the assay systemdescribed therein is based on antibodies that recognize epitopes in theA-beta polypeptide between amino acids 1 to 28 of A-beta, and does notdifferentiate between A-beta (1-40) and A-beta (1-42). In addition, theassay is not anticipated to be sufficiently sensitive to detect thelevels of A-beta (1-42) that are expected to accumulate in beta-APPtransfected mammalian cells in the 96-well microtiter plate formatrequired for high-throughput drug screening.

While mutations have been introduced within the A-beta sequence ofbeta-APP (for example Citron M, Teplow D B, Selkoe D J 1995 Neuron14:661-670), the use of beta-APP molecules epitope-tagged within theA-beta sequence has not been explored.

SUMMARY OF THE INVENTION

The present invention provides methods that enable one skilled in theart to identify and characterize beta amyloid production inhibitors.Recombinant expression constructs were prepared containing an epitopefor a high-affinity antibody centered about the alpha secretase cleavagesite of the A-beta fragment of the beta-APP. Cell lines were establishedthat express these mutant forms of beta-APP and secrete fragments ofepitope-tagged beta-APP. In addition, epitope-tagged beta amyloidfragments were also prepared by chemical synthesis.

An object of the invention provides assays and methods for the detectionand characterization of epitope-tagged beta-APP and fragments thereof.The invention provides enzyme-linked immunosorbent assays (ELISA) andother immunological methods suitable for the detection of alpha, beta,and gamma secretase modulators in cell-based and cell-free assaysystems. An accumulation of epitope-tagged A-beta was observed in cellstransfected with the expression cassette. The cultured cells wereexposed to test compounds that cause a change in the secreted amount ofsoluble epitope tagged fragments of beta-APP. The present assays andmethods may be used to identify beta amyloid precursor processingmodulators and to develop therapeutic modalities based on modificationof beta amyloid precursor processing.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Shows a possible location of an epitope tag in the A-betasequence of the beta-APP and predicted accumulation of epitope taggedcleavage fragments. The A-beta fragment (1-42), with the proposedproteolytic cleavage sites for secretases (alpha-, beta-, gamma 1 [40]-,and gamma 2 [42]), is indicated. The epitope tag in this example iscentered on the alpha secretase site (amino acids 16 to 17 in A-beta).Cleavage by beta and gamma secretases is expected to lead to anaccumulation of epitope tagged A-beta (1-40) and A-beta (1-42) in theconditioned medium, whereas cleavage by alpha secretase (within theepitope tag) is expected to destroy or reduce the accumulation ofepitope tagged A-beta fragments in the conditioned medium.

FIG. 2 Shows an immunoblot analysis of HEK 293 (human embryonic kidneycell line, ATTC #CRL-1573) cell lysates after transfection withepitope-tagged beta-APP. Cell lysates were prepared by lysis of HEK 293cells into SDS and were fractionated by SDS-PAGE, followed by transferto nitrocellulose membranes. The membranes were developed with mAB 22C11(epitope in the N-terminus of full-length beta-APP; lanes 1 and 2), mABanti-HA 11 (influenza hemagglutinin epitope: YPYDVPDYA) (SEQ ID NO: 6)(directed to the HA 11 epitope tag; lanes 3 and 4), and mAB 9E10(directed to the myc epitope tag; lanes 5 and 6). Lane 1, HEK 293 cellstransfected with HA 11 beta-APP 695; lane 2, HEK 293 cells transfectedwith vector alone (‘Mock-transfection’); lane 3, HEK 293 cellstransfected with HA 11 beta-APP 695; lane 4, HEK 293 cells transfectedwith vector alone; lane 5, HEK 293 cells transfected with myc beta-APP695; lane 6, HEK 293 cells transfected with vector alone. The relativemobility of molecular weight standards is indicated to the left.

FIG. 3 Shows an accumulation of beta-APP fragments into HEK 293conditioned medium. The 24 hour serum-free conditioned medium (lanes 1and 2) or cell lysates (lanes 3 and 4) of HEK 293 cells transfected withvector alone (lanes 1 and 3) or HA 11 beta-APP 695 (lanes 2 and 4) wereharvested. The resulting polypeptides were fractionated by SDS-PAGE (10%acrylamide in separating gel) and transferred to nitrocellulosemembranes. Panel A was developed with mAB anti-HA 11, whereas panel Bwas developed with mAB 22C11. The relative mobility of molecular weightstandards is indicated to the right.

FIG. 4 Shows the detection of epitope-tagged beta-APP fragments in HEK293 conditioned medium after transfection with HA 11 beta-APP 695.

Panel A: Microtiter wells were coated with mAB anti-HA 11 and afterblocking, incubated with a dose-response of a synthetic HA 11 A-beta(1-40) peptide containing the HA 11 epitope centered on the alphasecretase cleavage site. Bound A-beta HA 11 was detected with polyclonalantibodies specific for position 1 (Serotec) or position 40 (QCB),followed by HRP-labeled anti-rabbit IgG and TMB substrate. The change ofabsorbance at 650 nM was monitored and results are corrected for bindingof secondary antibodies to wells not incubated with the A-beta HA 11peptide. Results are expressed as change of absorbance per minute(mOD/minute).

Panel B: Microtiter wells were coated as in panel A and incubated withthe indicated dilutions of HEK 293/HA 11 beta-APP 695 conditioned medium(24 hours). Bound HA 11 beta-APP 695 fragments were detected withantibodies specific for position 1 and 40 as in panel A. Results areexpressed and corrected as in panel A.

FIG. 5 Shows a time-course of the accumulation of HA 11 A-beta (1-40)and A-beta (1-42) in HEK 293/HA 11 beta-APP 695 conditioned medium. HEK293/HA 11 beta-APP 695 was cultured in serum-free medium containing 0.2%bovine serum albumin in 96-well microtiter plates for the indicated timeintervals. The accumulation of HA 11 A-beta (1-40) and A-beta (1-42) wasdetermined. For HA 11 A-beta polypeptides ending at position 40,microtiter wells were coated with mAB anti-HA 11 and bound polypeptideswere detected with rabbit anti-A-beta 40 (QCB), followed by HRP-labeledanti-rabbit IgG. For the position 42-specific ELISA, microtiter wellswere coated with mAB anti-HA 11, and bound polypeptides were detectedwith biotin-labeled mAB 108 (position 42-specific), followed bystreptavidin-HRP conjugate. Results are corrected for binding ofsecondary antibodies in the absence of conditioned medium and expressedas change of absorbance at 650 nM per minute (mOD/minute).

FIG. 6 Shows the effect of MDL 28170 and Brefeldin A on the accumulationof HA 11 A-beta (1-40) in HEK 293/HA 11 beta-APP 695 conditioned medium.HEK 293/HA 11 beta-APP 695 cells were plated at confluence in 96-wellplates and the indicated dose-response of either MDL 28170 (panel A), orBrefeldin A (panel B) was added for 16 hours. The accumulation of HA 11A-beta (1-40) (position 40-specific antibody; QCB) was determined as inFIG. 5. Results are expressed as percentage inhibition of HA 11 A-beta(1-40) accumulation in comparison to wells incubated with vehicle(dimethyl sulfoxide, DMSO) alone.

FIG. 7 Shows an isolation of HA 11 A-beta from HEK 293/HA 11 beta-APP695 cells. Conditioned medium (serum-free containing 0.2% BSA) waspassed over an mAB anti-HA 11 affinity matrix. After washing, the columnwas eluted with 5% formic acid in water. The peak fractions were pooled,dried in a Speed-Vac, resuspended in water and the pH was adjusted to7.4 with Tris.

Panel A: The starting material, flow-through, and the pooled elutionfractions (after dilution to account for the concentration of the HA 11A-beta on the column) were analyzed by ELISA specific for position 40 inHA 11 A-beta as in FIGS. 4 and 5.

Panel B: The indicated dilutions of the pooled elution fractions wereanalyzed by ELISA specific for position 1, 40, and 42 in HA 11 A-beta.Note that approximately equal immunoreactivity is present for theposition 1 and 40 antibodies, whereas the 42-specific reactivity is lostwith 10-fold lesser dilution.

Panel C: The elution fractions were analyzed by SDS-PAGE (16.5%polyacrylamide in separating gel), followed by immunoblotting with mABanti-HA 11, followed by HRP-labeled anti-mouse Ig, and chemiluminescencedetection (ECL™, Amersham). Lane 1, elution fraction, stained with mABanti-HA 11; lane 2, elution fraction spiked with HA 11 A-beta peptide(50 ng); lane 3, purified A-beta HA 11 1-40 peptide; and lane 4, elutionfraction, stained under omission of anti-HA 11.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention generally employs conventionaltechniques of molecular biology, microbiology, recombinant DNA, andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature. See for example J. Sambrook et al.,“Molecular Cloning; A Laboratory Manual” (1989); “DNA Cloning”, Vol. Iand II (D. N. Glover ed., 1985); “Oligonucleotide Synthesis” (M. J. Gaited., 1984); “Nucleic Acid Hybridization” (B. D. Hames and S. J. Higginseds., 1984); “Transcription and Translation” (B. D. Hames & S. J.Higgins eds., 1984); “Animal Cell Culture” (R. I. Freshney ed., 1986);“Immobilized Cells and Enzymes” (IRL Press, 1986); “A Practical Guide toMolecular Cloning” (B. Perbal, 1984); the series, “Methods inEnzymology” (Academic Press, Inc.); “Gene Transfer Vectors for MammalianCells” (J. H. Miller and M. P. Calos eds., 1987, Cold Spring HarborLaboratory); Meth. Enzymol. (1987) 154 and 155 (Wu and Grossman, and Wueds., respectively); “Immunochemical Methods in Cell and MolecularBiology” (Academic Press, London); “Protein Purification: Principles andPractice”, Third Ed. (Scopes, Springer-Verlag, N.Y., 1994); and“Handbook of Experimental Immunology”, Volumes I-IV (Weir and Blackwell,eds., 1986).

The present invention results from the discovery that the A-betasequence of the beta-APP can be modified by inclusion of an epitope tagfor a specific monoclonal antibody without apparent loss of proteolyticcleavage by secretases and/or related enzyme systems. In particular, ithas been found that such epitope-tagged A-beta peptides are generated bycultured mammalian cells and may be measured in the conditioned medium.

A preferred embodiment of the invention provides methods andcompositions for the detection of epitope-tagged beta-APP processingmodulators useful for the treatment of diseases associated with alteredmetabolism of beta-APP. The method relies on the measurement of a verylow concentration in a fluid sample, typically in the range from 0.05ng/mL to 10 ng/mL, with the present invention providing highly sensitiveand specific methods for performing such measurements. In particular,detection methods of the present invention provide for measurement of HA11 A-beta at concentrations of 0.1 ng/mL and below, and are sufficientlysensitive and specific to distinguish HA 11 A-beta polypeptides with aC-terminal ending at position 40 or 42. This discovery is exemplified bythe detection of both HA 11 A-beta 40 and 42 in a 96-well tissue cultureformat. In addition, the suitability of the invention for theidentification of secretase inhibitors is exemplified by the inhibitionof HA 11 A-beta 40 accumulation in the presence of a reported secretaseinhibitor (MDL 28170; Mehdi S, Angelastro M R, Wiseman J S, Bey P, 1988Biochem Biophys Res Commun 157, 1117-1123), and agents that interferewith the secretory pathway (Brefeldin A). Thus, these examples indicatethat the assays of the invention are suitable for use in high-throughputscreening for the detection of secretase inhibitors.

The terms and abbreviations used herein have their normal meaningsunless otherwise designated. For example, “° C.” refers to degreesCelsius; “N” refers to normal or normality; “mmole” refers to millimoleor millimoles; “g” refers to gram or grams; and “M” refers to molar ormolarity.

All amino acid or protein sequences, unless otherwise designated, arewritten commencing from the amino terminus (“N-terminus”) and concludingwith the carboxy terminus (“C-terminus”).

All nucleic acid sequences, unless otherwise designated, are written inthe direction from the 5′ end to the 3′ end, frequently referred to as“5′ to 3′”. The abbreviations A,C,G, and T correspond to the5′-monophosphate forms of the deoxyribonucleosides deoxyadenine,deoxycytidine, deoxyguanine, and deoxythimine respectively, when theyoccur in DNA molecules.

The term “digestion” or “restriction” of DNA refers to the catalyticcleavage of the DNA with a restriction enzyme that acts only at certainsequences in the DNA molecule. The various restriction enzymes usedherein are commercially available and their reaction conditions,cofactors, and other requirements were used as would be known to one ofordinary skill in the art.

The term “plasmid” refers to an extrachromosomal (usually)self-replicating genetic element. The starting plasmids herein areeither commercially available, publicly available on an unrestrictedbasis, or can be constructed from available plasmids in accordance withpublished procedures. In addition, equivalent plasmids to thosedescribed are known in the art and will be apparent to the ordinarilyskilled artisan.

The term “expression vector”, as used herein, refers to any recombinantDNA cloning vector in which a promoter has been incorporated.

The term “promoter” refers to a DNA sequence that directs transcriptionof DNA to RNA.

The term “transcription”, as used herein, refers to the process wherebyinformation contained in a nucleotide sequence of DNA is transferred toa complementary RNA sequence.

The term “transfection”, as used herein, refers to the taking up of anexpression vector by a host cell, whether or not any coding sequencesare in fact expressed. Numerous methods for transfection are known tothe ordinarily skilled artisan, for example, calcium phosphateco-precipitation, lipofection, and electroporation. Successfultransfection is generally recognized when any indication of theoperation of this vector occurs within the host cell. Other routes ofproduction are well known to skilled artisans. In addition to theplasmid described, it is well known in the art that some viruses arealso appropriate vectors.

The term “neo-epitope”, as used herein, refers to an antibody bindingsite (epitope) that is only weakly or not expressed in the intactpolypeptide chain. Exo- and/or endoproteolytic cleavage of thepolypeptide chain increases the affinity of the antibody for the epitopeon the polypeptide chain. “Epitope tag” refers to an epitope for anantibody that is not naturally present in a polypeptide chain. Ittypically contains a defined polypeptide sequence of from about 5 toabout 15 amino acids that can be inserted into a naturally occurring orotherwise generated larger polypeptide.

The term “polyclonal antibody”, as used herein, refers to a populationof heterogeneous antibodies derived from multiple clones, each of whichis specific for one of a number of determinants found on an antigen.Serum obtained from immunizing suitable vertebrate hosts withpolypeptide sequences. “Monoclonal antibody” refers to antibodiesderived from an immortalized cell line capable of producing antibodieshaving desired specificity. “Capture antibody”, as used herein, refersto an antibody that is linked to a solid-phase matrix in a biologicallyactive form. A number of procedures for the immobilization of antibodiesare known in the art, including passive absorption to plastic surfacesand chemical cross-linking. “Binding substance”, as used herein, refersto an antibody capable of binding to an epitope in a polypeptide chain.

The term “solid-phase”, as used herein, refers to a surface that iscapable of binding a polypeptide chain. A number of solid-phase surfacesare known in the art including, but not limited to, plastic surfaces andagarose beads.

As used herein, the term “recombinant expression system” refers to astrong promoter encoding sequence, a strong ribosome binding site, a DNAsequence encoding a naturally-occurring or otherwise generated DNAsequence, a selectable marker encoding sequence, and a poly-adenylationsignal encoding sequence. Upon transferring this recombinant expressionsystem into a suitable organism, it drives the production of a desiredprotein molecule. “Recombinant expression construct”, as used herein, isany DNA, whether naturally-occurring or otherwise from any source thatis capable of being inserted into any organism and drives the productionof a desired protein molecule over a desired time frame.

The term “cDNA construct”, as used herein, refers to a nucleic acidsequence, whether naturally-occurring or otherwise, from any sourcesthan can be replicated in a desired organism.

The terms “A-beta 40, A-beta 42”, as used herein, refer to a polypeptidefragment of beta APP which contains all or parts of sequence ID NO:1with a c-terminal ending at position 40 (Val-Gly-Gly-Val-Val-COOH), orending at position 42 (Val-Gly-Gly-Val-Val-Ile-Ala-COOH).

The term “A-beta”, as used herein, refers to an amino acid fragment,approximately 39 to 43 amino acids in length, of a large transmembraneglycoprotein referred herein as beta-APP. Beta-APP is encoded by a geneon the long arm of human chromosome 21. The 43-amino acid sequence ofA-beta is listed below:

NH₂-ASP ALA GLU PHE ARG HIS ASP SER GLY TYR GLU VAL HIS HIS GLN LYS LEUVAL PHE PHE ALA GLU ASP VAL GLY SER ASN LYS GLY ALA ILE ILE GLY LEU METVAL GLY GLY VAL VAL ILE ALA THR-COOH (SEQ ID NO:1).

The term “epitope-tagged A-beta”, as used herein, refers to apolypeptide sequence containing the above sequence (sequence ID NO:1)with the substitution and/or insertion of specific amino acidscomprising the epitope (i.e., antibody recognition site) of ahigh-affinity antibody.

The term “beta-APP” refers to a polypeptide that is encoded by a gene ofthe same name localized in humans on the long arm of chromosome 21, andwhich includes the A-beta sequence within its C-terminal regionsequence. Beta-APP is a single membrane-spanning, glycosylated proteinexpressed in a number of mammalian cells. Examples of specific isoformsof beta-APP that are currently known to exist in humans are the 695amino acid polypeptide (Kang J, Lemaire H, Unterbeck A, Salbaum J M,Masters C L et al 1987 Nature 325: 733-36), the 751 amino acid formdescribed by Ponte et al 1988 (Nature 331: 525-27), and the 770 aminoacid polypeptide described by Kitaguchi et al 1988 (Nature 331: 530-32).Examples of specific variants of beta-APP include point mutations thatcan differ in both position and phenotype (reviewed in: Selkoe D J 1994Annu Rev Cell Biol 10: 373-403). The skilled artisan will recognize thatthe epitope tagged A-beta sequence can be included in any of thesesplice variants or mutated beta-APP molecules.

The term “beta-APP fragment”, as used herein, refers to fragments ofbeta-APP other than those that solely consist of A-beta (i.e., sequenceID NO:1). Beta-APP fragments include amino acid sequences of beta-APP inaddition to those that comprise the A-beta polypeptide.

The term “conditioned media” refers to the aqueous extracellular fluidthat surrounds cells grown in tissue culture (in vitro) and consists of,but is not limited to, proteins, polypeptides, and amino acids secretedby the cells.

The term “body fluid” refers to those fluids of a mammalian host thatare expected to contain measurable amounts of epitope tagged A-beta orbeta-APP fragments and include, but are not limited to, cerebrospinalfluid, blood, peritoneal fluid, and urine.

The term “blood”, as used herein, includes but is not limited to, wholeblood, plasma, serum, and the cellular components present in blood.

The term “small molecule”, as used herein, refers to chemicalcompositions having a molecular weight less than approximately 900g/mole.

The term “biological polymer”, as used herein, refers to a macromoleculeoccurring in a living organism including, but not limited to, peptides,proteins, polysaccharides, or nucleic acids.

The term “splice variant”, as used herein, refers mRNA molecules thathave variant sequences due to the phenomenon of alternative splicing.Alternative splicing occurs when some introns of certain genes are notspliced out of some of the RNA molecules, leaving new combinations ofexons. Splice variants also include mRNA molecules whose variantsequences arise from splicing that occurs within the exon. Whendifferent splicing possibilities exist at several positions of thetranscript, a single gene can produce dozens of different proteins.

The term “production modulator”, as used herein, refers to compoundswhich inhibit A-beta peptide release and/or its synthesis, andaccordingly, have utility in treating Alzheimer's Disease.

The term “processing activities”, as used herein, refers to cellularactivity that results in the formation and/or release of A-betapeptides.

The term “competitive binding”, as used herein, refers to a situation inwhich one substance competes with another substance for a binding siteon a third substance.

Skilled artisans will recognize that the proteins of the presentinvention can be synthesized by a number of different methods. All ofthe amino acid compounds of the invention can be made by chemicalmethods well known in the art, including solid-phase peptide synthesis,or recombinant methods.

Practitioners of this invention realize that, in addition to the abovementioned expression systems, the cloned cDNA may be utilized in theproduction of transgenic animals, usually a mouse, in which expressionor overexpression of the proteins of the present invention can beassessed.

Skilled artisans will recognize that the function of the amino acidcomposition will not be affected by alterations in the epitope-taggedA-beta sequence. For example, some hydrophobic amino acids may beexchanged for other hydrophobic amino acids. Those altered amino acidcompositions that confer substantially the same function insubstantially the same manner as the exemplified amino acid compositionare also encompassed within the present invention. Although a preferredembodiment of this invention indicates the location of the epitope tagis centered around the alpha secretase cleavage site of A-beta, theskilled artisan will recognize that the epitope tag may also be placedin other locations in A-beta (1-42), or the 20 amino acids flankingeither of the N-terminal or C-terminal regions. Furthermore, it is wellknown in the art that besides the HA 11 epitope used in this invention,a number of different epitope tags may be used. Examples include theinclusion of the myc epitope and the flag epitopes.

According to the present invention, epitope-tagged A-beta and beta-APPfragments may be detected and quantified in a variety of biologicalsamples. Biological samples also include in vitro samples such asconditioned medium from cultured cells, transfected cell lines, andendogenous cell lines derived from transgenic animals. In vivo samples(i.e., body fluids) derived from transgenic animals may also beanalyzed. Detection and quantification may be accomplished by anytechnique capable of distinguishing epitope-tagged A-beta, or fragmentsthereof, from other beta-APP fragments that can be found in the sample.Immunological techniques can be employed using binding substancesspecific for epitope-tagged A-beta and other sequences within A-beta,including but not limited to antibodies, antibody fragments, andrecombinant antibodies, which bind specifically, and with highsensitivity, to epitope-tagged A-beta. Particularly suitable detectiontechniques include, but are not limited to, ELISA, immunoblotting, andradioimmunoassay (RIA).

A preferred immunoassay technique of the present invention is a two-siteor “sandwich” assay. This assay utilizes an epitope tag specificantibody and a second antibody that binds to an epitope other than thatbound by the first capturing antibody. The skilled artisan knows thatthe order of capturing and detecting antibody may be changed, and thatone of the antibodies may be labeled with radioisotopes, enzymes,biotin, streptavidin, or a similar substituent. Alternatively, ifdifferent immunoglobulin fragments or species for antibody productionwere utilized for the generation of antibodies, secondary labeledantibodies specific for the detection antibody may be used.Illustrations of these approaches are described in the Example section.

In the present invention immunological techniques may be combined withphysical separation methods. For example, gel electrophoresis may beused to separate complex biological samples containing epitope-taggedbeta-APP molecules, and/or fragments thereof, by size and/or chargedifferences. The gels may then be probed by immunoblotting or a similartechnique to identify specific polypeptides based on size or charge.This approach is illustrated in the following Example section.

A preferred embodiment of the present invention is cell lines capable ofexpressing epitope-tagged beta-APP, or fragments thereof, for use indrug screening. These may include some of the normal beta-APP isoforms(for example, of 695, 751, and 770 amino acids) or some of the familialvariants of beta-APP (e.g., Swedish and/or London mutation, reviewed inSelkoe D J 1994 Annu Rev Cell Biol 10: 373-403). In addition, in vivomonitoring of epitope-tagged A-beta, beta-APP fragments, or intactepitope-tagged beta-APP may be employed using animal models that harbora transgene. An especially preferred cell line employed in thisinvention is the widely available cell line HEK 293 (ATTC). A widevariety of vectors exist for the transformation of such mammalian hostcells, but the specific vector used herein is in no way intended tolimit the scope of the present invention. In addition to prokaryotes andmammalian host cells, eukaryotic microbes such as yeast cultures may beused.

An embodiment of the present invention provides cDNA construct thatencodes for beta-APP or fragments thereof, containing an epitope tagwithin the A-beta sequence or the immediate flanking regions of theA-beta sequence.

Another embodiment of the present invention provides an epitope-taggedbeta-APP or fragments thereof, derived from chemical synthesis orrecombinant expression systems.

Another embodiment of the present invention provides for a method fordetection of epitope-tagged A-beta peptide in a fluid sample in thepresence or absence of beta-APP or fragments thereof, comprising: (a)capturing a soluble A-beta from the sample using a first bindingsubstance that is a binding substance to the epitope tag; and (b) usinga labeled second binding substance which binds to an epitope on a secondregion of the soluble A-beta other than the epitope on the A-beta whichis bound by the first binding substance. In a preferred embodiment, thefirst binding substance is an epitope in A-beta distinct from theepitope tag and the second binding substance to the epitope tag. In amore preferred embodiment the competitive binding of a sample isdetermined between the first binding substance that detects the epitopetag in A-beta, and labeled epitope tagged beta-APP or fragments thereof.In a more preferred embodiment the soluble epitope-tagged A-beta iscaptured on a solid phase, and the capture is detected by exposing thesolid phase to the labeled second binding substance to the epitope tagor other sequences in A-beta and thereafter detecting the presence ofthe label on the solid phase. In a more preferred embodiment the epitopetag is centered at the site between A-beta amino acid residues 16 and 17and is a target to proteolytic cleavage and is substantially free fromcross-reactivity with beta-APP and fragments thereof other than A-beta.In a more preferred embodiment the binding substances are monoclonal orpolyclonal antibodies. In a more preferred embodiment, one of thebinding substances is specific for neo-epitope(s) generated in theN-terminus of A-beta upon action of beta secretase(s). In a furtherpreferred embodiment, one of the binding substances is specific forneo-epitope(s) generated in the C-terminus of A-beta upon action ofgamma secretase(s) and can differentiate between A-beta 40 and A-beta42. In a further preferred embodiment the epitope tagged beta amyloidprecursor fragments are present in culture medium, or blood, cerebralspinal fluid, urine, peritoneal fluid, or tissue extracts of organismharboring the epitope tagged beta-APP.

Another embodiment provides a method of screening compounds to identifyA-beta production modulators, comprising: (a) culturing cells expressingepitope tagged beta-APP or fragments thereof under conditions whichresult in secretion of a soluble epitope-tagged fragment of beta-APP;(b) exposing the cells to a test compound; and (c) detecting the amountof the soluble epitope-tagged beta-APP fragments present in solution,wherein an altering in the amount of the soluble beta-APP fragment insolution is compared to the amount of soluble beta-APP fragment presentwhen the cells are not exposed to the compound indicates that thecompound is an A-beta production modulator. In a preferred embodimentthe cells are cultured human embryonic kidney 293 cells or derived froman organism expressing epitope tagged beta-APP or fragments thereof. Ina preferred embodiment, the test compound is exposed at a concentrationfrom 1 ρM to 1 mM. In a preferred embodiment the test compound is asmall molecule. In a preferred embodiment the test compound is abiological polymer. In a further preferred embodiment, the assay candetect specific inhibitors of beta-APP at position 1, 40, and 42 of theA-beta sequence.

Another embodiment of the present invention provides an in vitro methodto identify beta amyloid processing activities, comprising: (a)incubation of epitope-tagged beta-APP or fragments thereof with a sourceof processing activities; and (b) detecting the amount of proteolyticprocessing by methods as outlined above. In a preferred embodiment, themethod is employed to enrich or purify beta amyloid processingactivities. In a preferred embodiment, the source of processingactivities is cultured cells, tissue or tissue extracts.

Another embodiment of the present invention provides an in vitro methodto identify beta amyloid processing activities, comprising: (a)incubation of epitope-tagged beta-APP or fragments thereof with a sourceof processing activities; and(b) detecting the amount of proteolyticprocessing by immunoblotting.

A preferred embodiment of the above methods is where the epitope tag islocated within mutant forms of beta-APP or splice variants of beta-APP.

Preferred embodiments of the invention were chosen for the purpose ofillustration and description, but are not intended in any way torestrict the scope of the invention.

The invention can be further understood by the following examples inwhich parts and percentages are by weight unless otherwise indicated.The examples presented below are provided as a guide to the practitionerof ordinary skill in the art, and are not to be construed as limitingthe invention in any way.

EXAMPLE 1

Location of an Epitope Tag in the A-beta Sequence of the Beta-APP andExpected/Predicted Accumulation of Epitope-tagged Beta-APP CleavageFragments

The sequence of A-beta (1-42) is indicated in FIG. 1. A possiblelocation of an epitope tag within the A-beta sequence of the beta-APP isalso shown in FIG. 1. In this example, the epitope tag is centered onthe alpha secretase cleavage site (indicated by alpha). In addition, thecleavage sites for beta secretase (indicated by beta, generally cleavedbetween −1/+1 of A-beta), and gamma 1 secretase (indicated by gamma 1,generally cleaved between 40/41) and gamma 2 secretase (indicated bygamma 2, generally cleaved between 42/43) are indicated. The numberingrefers to the polypeptide sequence of the wild type A-beta polypeptide(i.e., position 1, D; position 2, A; position 3, E; position 40, V;position 41, I; position 42, A). Alternative positions of the epitopetag(s) may be throughout the A-beta sequence so long as the epitope tagdoes not interfere with the cellular processing of beta-APP. Thepredicted accumulation of epitope-tagged beta amyloid cleavage fragmentswith a location of an epitope tag centered on the alpha secretase site(generally cleaved between amino acids 16 and 17 of A-beta) isindicated. More specifically, cleavage by beta and gamma secretases isexpected to result in the accumulation of epitope-tagged A-beta, whereascleavage within the epitope tag by proteolytic enzyme system(s) shouldreduce the expression or destroy the epitope tag. An epitope tag may beintroduced by replacing of existing amino acids in A-beta, by insertionof additional amino acids, or by a combination of both.

EXAMPLE 2

Immunoblotting Analysis of HEK 293 Cell Lysates After Transfection withEpitope-tagged Beta-APP

Generation of cDNA constructs: Site-directed mutagenesis was utilized toincorporate the cDNA sequence for either the myc or HA 11 epitope tagwithin the A-beta fragment of the beta-APP (695 isoform). Morespecifically, the 5′ primer contained the unique EcoRI sequence,followed by A-beta sequences and the epitope tag, whereas the 3′ primercontained the 3′ end of the beta-APP reading frame, followed by a stopcodon and a XbaI cloning site. The following PCR primers were utilized:myc epitope, 5′ primer: 5′ GAT GCA GAA TTC CGA CAT GAC TCA GGA TAT GAAGAA CAA AAA CTC ATT TCA GAA GAA GAT CTC GAA GAT GTG GGT TCA AAC AAA GGTGC 3′ (SEQ.ID.NO.:2) (mutated sequences underlined); HA 11 epitope, 5′primer: 5′ GAT GCA GAA TTC CGA CAT GAC TCA GGA TAT GAA GTT TAT CCA TATGAT GTG CCA GAT TAT GCA GAA GAT GTG GGT TCA AAC AAA GGT GC 3′(SEQ.ID.NO.:3) (mutated sequences underlined); 3′ primer (for bothconstructs): 5′ AGC TTC TAG AGG TCT AGT TCT GCA TCT GCT CAA 3′(SEQ.ID.NO.:4). Using the full-length beta-APP cDNA (695 amino acids inlength) as a template, the DNA fragment was amplified,restriction-digested with EcoRI and XbaI and subcloned into pUC 18. The5′ XbaI/EcoRI fragment of beta-APP was gel-isolated after restrictiondigestion and also subcloned into pUC 18. The full-length reading frameof the epitope-tagged beta-APP cDNA was assembled into pUC 18 from theXbaI/EcoRI 5′ fragment and EcoRI/XbaI 3′ fragment. The XbaI/XbaIfragment containing the beta-APP 695 cDNA was transferred into PM3AR, aneukaryotic expression vector under control of the CMV virus promotercontaining the EBV oriP for episomal maintenance of the plasmid. Thepresence of the desired mutations was confirmed by double-stranded DNAsequencing. In addition, the myc and HA 11 epitope tags were introducedinto the Swedish beta-APP mutant.

Culture of HEK 293 E and Transfection

HEK 293 E cells were maintained in DMEM (Gibco-BRL) supplemented with10% fetal bovine serum (Hyclone FBS), 25 μg/mL penicillin, 25 μg/mLstreptomycin (Gibco-BRL), and 250 μg/mL G 418 (Gibco-BRL; geneticin).Cells were transfected with the lipofectin method according to themanufacturer (Gibco-BRL Lipofectamine). One day after transfection, theconditioned medium was replaced with selective medium (media as abovecontaining in addition 250 μg/mL hygromycin B (Boehringer Mannheim)).

Immunoblotting Analysis of HEK 293 Cell Lysates

Confluent culture of HEK 293 cells (1×10⁶ cells/cm²) transfected withvector control (“mock-transfection”, lanes 2, 4, and 6), myc beta-APP695 (lane 5), or HA 11 beta-APP 695 (lanes 1 and 3) were washed withPBS, and cell lysates prepared by the addition of SDS sample buffercontaining 50 mM dithiothreitol. The samples were boiled, fractionatedby SDS-PAGE (10% acrylamide in the separating gel), and transferred tonitrocellulose membranes. The membranes were blocked (PBS containing 5%dry milk) and washed extensively with PBS containing 0.1% Tween-20. Themembranes were incubated with mAB 22C11 (1 μg/mL in PBS/0.1% Tween-20;Boehringer Mannheim, lanes 1 and 2), mAB anti-HA 11 (1:1000 dilution ofascites in PBS/0.1% Tween-20; BABCO, lanes 3 and 4), or mAB anti-myc(clone 9E10, 1 μg/mL in PBS/0.1% Tween-20, Calbiochem, lanes 5 and 6)for 1 hour at room temperature. The membranes were washed with PBS/0.1%Tween-20, followed by incubation with sheep anti-mouse Ig (1:10,000dilution, Amersham) and extensive washing in PBS/0.1% Tween-20. Themembranes were incubated with ECL™ chemiluminescence reagent (Amersham)and exposed to X-ray film.

EXAMPLE 3

Accumulation of Beta-APP Fragments in HEK 293/HA 11 Beta-APP 695Conditioned Medium

The 24 hour conditioned medium (serum-free, 1×10⁶ cells/cm²) of HEK 293cells transfected with vector alone (lane 1) or 293/HA 11 beta-APP 695cells (lane 2) was harvested, and cell lysates derived from vector alone(lane 3) or HA 11 beta-APP 695 (lane 4) were prepared as in Example 2.The resulting polypeptides were fractionated by SDS-PAGE (10% acrylamidein the separating gel) and transferred to nitrocellulose membranes.Panel A was probed with anti-HA 11 to detect epitope-tagged beta-APP orfragments thereof, whereas panel B was developed with mAB 22C11 todetect beta-APP or fragments thereof, which span essentially theN-terminus of full-length beta-APP (the epitope of mAB 22C11 is locatedin the first 100 amino acids of full-length beta-APP). Note the strongsignal in the conditioned medium using mAB 22C11 (panel B, lane 2),whereas little or no staining was obtained with anti-HA 11 (panel A,lane 2). In contrast, similar staining intensities were obtained in thecell lysates using either antibody (compare panels A and B, lanes 4).Taken together, these results are consistent with cleavage of theepitope tag by alpha secretase-like activities in HEK 293 cells,resulting in reduction and/or loss of the HA 11 epitope.

EXAMPLE 4

Detection of Epitope-tagged Beta-APP Fragments in HEK 293 ConditionedMedium After Transfection with HA 11 Beta-APP

A synthetic HA 11 A-beta peptide with the following sequence wassynthesized: NH₂-ASP ALA GLU PHE ARG HIS ASP SER GLY TYR GLU GLU GLN LYSLEU ILE SER GLU GLU ASP ILE GLU ASP VAL GLY SER ASN LYS GLY ALA ILE ILEGLY LEU MET VAL GLY GLY VAL VAL-COOH (SEQ.ID.NO.:5). In this peptide,the wild-type A-beta sequence is replaced, between amino acids 12 to 21,with the HA 11 epitope (i.e., TYR PRO TYR ASP VAL PRO ASP TYR ALA(SEQ.ID.NO.:6)). The resulting peptide was purified by HPLC to greaterthan 95% purity and dissolved in 50% DMSO/water, and stored at −80° C.prior to use.

Panel A: Immulon 2 microtiter wells were coated with mAB anti-HA 11 (5μg/mL in PBS, 4° C., overnight), washed with PBS and blocked with 5% BSAin PBS. The peptide was diluted in PBS/0.1% BSA/0.1% Tween-20 andincubated in the wells for 2 hours at room temperature. After washing(PBS), the wells were incubated with rabbit anti-A-beta position 1(Serotec; 5 μg/mL in PBS/0.1% BSA/0.1% Tween-20) or rabbit anti-A-betaposition 40 (QCB; 5 μg/mL in PBS/0.1% BSA/0.1% Tween-20) for 1 hour atroom temperature. After washing, the wells were incubated withHRP-labeled anti-rabbit IgG (Amersham; 1:1000 dilution in PBS/0.1%BSA/0.1% Tween-20) for 1 hour. The wells were washed again, andincubated with TMB substrate. The change of absorbance of duplicatewells was determined at 650 nM and results corrected for binding ofantibodies to wells in the absence of peptide. The signal obtained waslinear with respect to the HA 11 A-beta peptide concentration, andapproached saturation at 10 ng/mL peptide. The lower detection limit forthe position 40 ELISA was approximately 0.1 ng/mL HA 11 A-beta peptide.

Panel B: Confluent cultures of HEK 293/HA 11 beta-APP 695 cells wereincubated in serum-free medium containing 0.2% BSA for 16 hours. At theend of the incubation period, dilutions of the conditioned medium wereprepared (100%=undiluted; 10%=1:10 diluted, etc.) and HA 11 containingbeta-APP fragments were quantified as in panel A. The signal decreasedwith increasing dilution of the conditioned medium. Note that the ratiobetween the position 40 and position 1 signal is similar using thepurified HA 11 A-beta peptide and the conditioned medium. Thisobservation suggests that similar amounts of A-beta N- and C-termini arepresent in the conditioned medium and purified system.

EXAMPLE 5

Time-course of the Accumulation of HA 11 A-beta (1-40) and (1-42) in HEK293/HA 11 Beta-APP Conditioned Medium: The Sensitivity of the ELISASystem is Sufficient for a 96-well Format

HEK 293/HA 11 beta-APP 695 cells were plated in 96-well tissue culturedishes in serum-free medium containing 0.2% BSA (1×10⁶ cells/cm²). Theconditioned medium was harvested at the indicated times and analyzed forthe presence of HA 11 A-beta position 40, as in example 4. HA 11 A-betapolypeptides ending at position 42 were detected in a similar ELISAsystem. Briefly, microtiter wells were coated with anti-HA 11, incubatedwith the conditioned medium. Bound HA 11 A-beta 42 was detected withbiotin-labeled mAB 108 (specific for the neo-epitope generated uponcleavage of beta-APP by gamma 2 secretase), followed by HRP-labeledstreptavidin and TMB substrate. Note that the sensitivity of the ELISAfor position 40 and 42 is sufficient for a 96-well format, thus makingthese assays suitable for high-throughput screening. Using the purifiedHA 11 A-beta 1-40 peptide, the HA 11 A-beta peptide concentration in the24 hour conditioned medium was estimated to be 0.6 ng/mL.

EXAMPLE 6

Effect of MDL 28170 and Brefeldin A on the Accumulation ofEpitope-tagged Beta-APP Fragments in HEK 293/HA 11 Beta-APP ConditionedMedium

HEK 293/HA 11 beta-APP 695 cells were plated at confluency (1×10⁶cells/cm²) in 96-well tissue culture dishes in serum-free mediumcontaining 0.2% BSA in the presence of the indicated concentration ofeither a previously described gamma secretase inhibitor MDL 28170 (Mehdiet al 1988 Biochem Biophys Res commun 157: 1117-1123) (panel A) orBrefeldin A (panel B). After 16 hours, the conditioned medium washarvested and analyzed using the HA 11 A-beta position 40-specificELISA. Note the decrease in the HA 11 A-beta accumulation in thepresence of both brefeldin A and MDL 28170. This experiment points tothe suitability of the HA 11 beta-APP 695 cells for the detection ofmodulators of A-beta secretion/accumulation.

EXAMPLE 7

Isolation of HA 11 A-beta from HEK 293/HA 11 Beta-APP 695 Cells

HEK 293/HA 11 beta-APP 695 cells (1×10⁶ cells/cm²) were grown toconfluency and placed in serum-free medium containing 0.2% BSA for 16hours. The conditioned medium was harvested, cellular debris removed bycentrifugation at 1,000 g for 10 minutes, and stored at −80° C. prior touse. mAB anti-HA 11 matrix (BABCO) was equilibrated in serum-free mediumcontaining 0.2% BSA, and the conditioned medium (4 L) was passed overthe column at a flow-rate of 100 mL/hour. The column was washed withserum-free medium containing 0.2% BSA, distilled water, and eluted with5% formic acid in distilled water. The elution fractions were pooled,dried in a speed vac, resuspended with water and neutralized with Tris.

The HA 11 A-beta concentration in the starting material, flow-through,and elution fraction (after dilution to account for the concentration onthe affinity column) was estimated by ELISA specific for position 40 asin Examples 4 and 5. Note that the ELISA immunoreactivity issignificantly reduced by absorption of the conditioned medium to theanti-HA 11 affinity matrix and that the epitope tagged A-beta fragmentscan be recovered from the column.

The indicated dilutions of the elution fraction were analyzed by ELISAspecific for position 1, 40, and 42 in HA 11 A-beta. Note that position1, 40, and 42 immunoreactivity is present in the elution fraction.Moreover, while the amount of 1 and 40 immunoreactivity appears to besimilar, the 42-signal is lost at a lower dilution of the conditionedmedium, suggesting that the concentration of HA 11 A-beta 42 isapproximately 10-fold lower than that ending at 40.

The elution fraction was fractionated by SDS-PAGE (10-20% Tris Tricinegradient gel), transferred to nitrocellulose membranes and analyzed byimmunoblotting using mAB anti-HA 11. Lane 1, elution fraction; lane 2,elution fraction spiked with 50 ng HA 11 A-beta peptide; lane 3,purified HA 11 A-beta peptide; lane 4, immunoblotting of the elutionfraction under omission of the primary antibody. Note that polypeptidesco-migrating with the purified HA 11 A-beta peptide standard are presentin the elution fraction.

6 1 43 PRT Human 1 Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val HisHis Gln Lys 1 5 10 15 Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn LysGly Ala Ile Ile 20 25 30 Gly Leu Met Val Gly Gly Val Val Ile Ala Thr 3540 2 89 DNA Human 2 gatgcagaat tccgacatga ctcaggatat gaagaacaaaaactcatttc agaagaagat 60 ctcgaagatg tgggttcaaa caaaggtgc 89 3 89 DNAHuman influenza virus 3 gatgcagaat tccgacatga ctcaggatat gaagtttatccatatgatgt gccagattat 60 gcagaagatg tgggttcaaa caaaggtgc 89 4 33 DNAHuman 4 agcttctaga ggtctagttc tgcatctgct caa 33 5 40 PRT Human 5 Asp AlaGlu Phe Arg His Asp Ser Gly Tyr Glu Glu Gln Lys Leu Ile 1 5 10 15 SerGlu Glu Asp Ile Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile 20 25 30 GlyLeu Met Val Gly Gly Val Val 35 40 6 9 PRT Human 6 Tyr Pro Tyr Asp ValPro Asp Tyr Ala 1 5

What is claimed is:
 1. A method of screening compounds to identifyA-beta production modulators, comprising: (a) culturing cells expressingepitope tagged beta-APP or fragments thereof, said beta-APP or beta-APPfragment containing an amino acid sequence of A-beta having an epitopetag within the A-beta amino acid sequence, under conditions which resultin secretion of a soluble epitope-tagged fragment of beta-APP; (b)exposing the cells to a test compound; and (c) detecting the amount ofthe soluble epitope-tagged beta-APP fragments present in solution,wherein an altering in the amount of the soluble beta-APP fragment insolution is compared to the amount of soluble beta-APP fragment presentwhen the cells are not exposed to the compound indicates that thecompound is an A-beta production modulator.
 2. A method according toclaim 1, wherein the cells are cultured human embryonic kidney 293 cellsor derived from an organism expressing epitope tagged beta-APP orfragments thereof.
 3. A method according to claim 1, wherein the testcompound is exposed at a concentration from 1 ρM to 1 mM.
 4. A methodaccording to claim 1, wherein the test compound is a small molecule. 5.A method according to claim 1, wherein the test compound is a biologicalpolymer.
 6. A method according to claim 1, wherein the assay can detectspecific inhibitors of beta-APP at position 1, 40, and 42 of the A-betaamino acid sequence.
 7. A method according to claim 1, wherein theepitope tag is located within the A-beta amino acid sequence of mutantforms of beta-APP or splice variants of beta-APP.